Although it is generally held that C-peptide, a pancreatic hormone that is co-secreted with insulin, does not possess biological activity of its own, recent work by our research team and others has raised doubts concerning this view. Although cumulative clinical and experimental evidence indicate that C-peptide replacement may have an impact on renal dysfunction in type I diabetes, the exact nature of its bioactivity, membrane interactions, and physiological effect on the renal microcirculation require further clarity. We plan to examine these aspects using a multi-faceted approach of in vitro biochemistry in isolated renal endothelial cells and innovative in vivo bioimaging of the renal cortical microcirculation, in which the influences of the native environment persist. Diabetics have an anomaly in redox regulation that precedes the development of chronic microvascular dysfunction. A potential explanation for this dysregulation rests with the interaction between defective vasoprotective systems and cellular redox status. Reduced vasoprotection as a consequence of compromised heme oxygenase (HO) activity could account for such an event. The ultimate function of this redox-sensitive system depends upon an adequate pool of NADPH. The major source of mammalian NADPH is glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway. During diabetes, G6PD activity is diminished, reducing the synthesis and bioavailability of endothelial NADPH. We believe that unraveling the mechanism(s) by which C-peptide augments NADPH synthesis and enhances HO-mediated vasoprotection will lead to fundamental insights into the renal microvascular pathogenesis of type I diabetes. We hypothesize that C-peptide improves renal endothelial NADPH thereby restoring vasoprotection in type I diabetes, and that a component of this C- peptide-mediated renal vasoprotection is related to re-establishing endothelial antioxidant potential. To address this we will: i) determine if C-peptide normalizes basal renal cortical microvascular function by restoring endogenous renal vasoprotection, ii) evaluate whether C-peptide improves basal renal cortical endothelial NADPH levels by re-establishing NADPH synthesis, and iii) determine if C-peptide reduces oxidant production in the renal cortical microcirculation. LAY SUMMARY: In addition to insulin, other pancreatic hormones are absent in type I diabetics and may help to mediate recovery from diabetic complications. We believe that one of these products, C-peptide, participates in important microvascular functional processes. We have already shown that C-peptide restores kidney microvascular function. We hope to determine how C-peptide interacts with other cellular factor(s) and processes to re-establish proper microvascular function in type I diabetics.